While we now have relatively complete lists of all proteases expressed in any given organism, our understanding of the functional roles of these enzymes, for the most part, remains largely unclear. The development of new technologies that will allow the imaging of protease activity is a critical step to begin to map out complex proteolytic cascades as well as to validate proteases as biomarkers for human disease. In the first phase of funding for this project we developed fluorescently labeled small molecule activity based probes (ABPs) that target two major classes of cystiene proteases, the cathepsins and caspases. We then demonstrated that fluorescent ABPs could be used for functional, non-invasive imaging studies of these proteases in various mouse models of human cancer. In the next phase of the project we will apply the probes from the initial funding period to mouse models of inflammation and atherosclerosis. We will also engineer several new classes of imaging probes that target proteases that play roles in the regulation of inflammation. These targets include cathepsins S and K, caspases-1, -4 and -5 and the immunoproteasome. We believe that these reagents will expand the use of the ABP technology platform to a large number of important human diseases that involve the process of inflammation.